Radiolabeled compounds for thrombus imaging

ABSTRACT

This invention relates to radiolabeled scintigraphic imaging agents, and methods and reagents for producing such agents. Specifically, the invention relates to specific binding compounds, including peptides, that bind to a platelet receptor that is the platelet GPIIb/IIIa receptor, methods and kits for making such compounds, and methods for using such compounds labeled with technetium-99m via a covalently-linked radiolabel-binding moiety to image thrombi in a mammalian body.

This application is a divisional of allowed U.S. patent application Ser.No. 08/253,317, filed Jun. 3, 1994 and now U.S. Pat. No. 5,830,856,which is a continuation-in-part of U.S. patent application Ser. No.08/044,825, filed Apr. 8, 1993 and now abandoned, which is acontinuation-in part of U.S. patent application Ser. No. 07/653,012,filed Feb. 8, 1991 and now abandoned; this application is also acontinuation-in part of allowed U.S. patent application Ser. No.08/335,832, filed Jan. 5, 1995 and now U.S. Pat. No. 5,925,331, is aU.S. national phase application claiming priority to PCT/US93/04794,filed May 21, 1993.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to scintigraphic imaging agents and reagents, andmethods for producing such agents and reagents. Specifically, theinvention relates to reagents that can be radiolabeled withtechnetium-99m (Tc-99m), methods and kits for making and radiolabelingsuch reagents, and methods for using such radiolabeled reagents to imagesites of thrombus formation in a mammalian body.

2. Description of the Prior Art

Thrombosis and thromboembolism, in particular deep vein thrombosis (DVT)and pulmonary embolism (PE), are common clinical conditions that areassociated with significant morbidity and mortality. It has beenestimated that in the U.S. approximately 5 million patients experienceone or more episodes of DVT per year and that over 500,000 cases ofpulmonary embolism occur, resulting in 100,000 deaths (J. Seabold,Society of Nuclear Medicine Annual Meeting 1990). It has also beenestimated that over 90% of all pulmonary emboli arise from DVT in thelower extremities. Anticoagulant therapy can effectively treat theseconditions if applied early enough. However, such treatment isassociated with risks (e.g. internal bleeding) that prevent unnecessaryprophylactic application. More advanced techniques of thrombolyticintervention (such as the administration of recombinant tissueplasminogen activator or streptokinase) can be used in acute cases, butthese techniques carry even greater risk. Moreover, effective clinicalapplication of these techniques requires that the site of the offendingthrombus be identified so as to monitor the effect of treatment.

For these reasons, a rapid means of localizing thrombi in vivo, mostpreferably using non-invasive methods, is highly desirable. Methodscurrently utilized for the identification of sites of deep-veinthrombosis are contrast venography and compression B-mode ultrasound;the choice of which technique is used depends on the expected locationof the thrombus. However, the former technique is invasive and bothtechniques are uncomfortable for the patient. In addition, these methodsare in many cases either unsuitable or yield inaccurate results.

Current methods used to diagnose PE include chest X-ray,electrocardiogram (EKG), arterial oxygen tension, perfusion andventilation lung scans, and pulmonary angiography. Apart from the latter(invasive) procedure, none of these methods is capable of providing anunequivocal diagnosis.

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ⁶⁸ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, and ¹⁶⁹ Ybe.Of these radionuclides, Tc-99m and ¹¹¹ In are preferred singlephoton-emitting radionuclides and ⁶⁸ Ga is preferred as apositron-emitting radionuclide. Tc-99m is a preferred radionuclidebecause it does not emit alpha or beta particle radiation and emitsgamma radiation at about 140 keV, has a physical half-life of 6 hours,and is readily available on-site using a molybdenum-99/technetium-99mgenerator.

A gamma-emitting radiotracer that binds specifically to a component of athrombus in preference to other tissues when administered in vivo canprovide an external scintigraphic image which defines the location ofthe thrombus-bound radiotracer and hence the thrombus. Thrombi areconstructs of blood cells (largely activated platelets) enmeshed incross-linked fibrin. Activated platelets are particularly good targetsfor radioimaging thrombi because they are not normally found incirculating blood (which contains unactivated platelets).

Activated platelets express the GPIIb/IIIa receptor on their cellsurfaces. The normal ligand for this receptor is fibrinogen (Plow etal., 1987, Perspectives in Inflammation, Neoplasia and Vascular CellBiology, pp. 267-275). However, small, synthetic analogues, which may bebut are not necessarily peptides, have been developed that bind to thisreceptor (examples include Klein et al., 1992, U.S. Pat. No. 5,086,069and Egbertson et al., 1992, European Patent Application No. EPA0478328A1). Although many of these synthetic molecules bind with onlylow affinity, others have been made that have very high affinity (seeEgbertson et al., ibid.).

Attempts to provide radiotracers for imaging thrombi are known in theprior art. These include autologous platelets, labeled with either ¹¹¹In or ^(99m) Tc (Tc-99m), and ¹²³ I- and ¹²⁵ I-labeled fibrinogen (thelatter detected with a gamma scintillation probe as opposed to a gammacamera). Additional radiolabeled compounds used to label thrombi includeplasmin, plasminogen activators, heparin, fibronectin, fibrin FragmentE₁ and anti-fibrin and anti-platelet monoclonal antibodies (see Knight,1990, Sem. Nucl. Med. 20: 52-67 for review).

Compounds having the ability to bind to the platelet GPIIb/IIIa receptorare known in the prior art.

Ruoslahti & Pierschbacher, U.S. Pat. No. 4,578,079 describe peptides ofsequence X-Arg-Gly-Asp-R-Y (SEQ ID NO: 1), wherein X and Y are either Hor an amino acid, and R is Thr or Cys, the peptides being capable ofbinding to platelets.

Ruoslahti & Pierschbacher, U.S. Pat. No. 4,792,525 describe peptides ofsequence Arg-Gly-Asp-X (SEQ ID NO: 2), wherein X is Ser, Thr or Cys, thepeptides being capable of binding to platelets.

Klein et al., 1992, U.S. Pat. No. 5,086,069 disclose guanine derivativesthat bind to the GPIIb/IIIa receptor.

Pierschbacher et al., 1989, PCT/US88/04403 discloseconformationally-restricted RGD-containing peptides for inhibiting cellattachment to a substratum.

Nutt et al., 1990, European Patent Application 90202015.5 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202030.4 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202031.2 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90202032.0 disclosecyclic RGD peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90311148.2 disclosecyclic peptides that are fibrinogen receptor antagonists.

Nutt et al., 1990, European Patent Application 90311151.6 disclosecyclic peptides that are fibrinogen receptor antagonists.

Ali et al., 1990, European Patent Application 90311537.6 disclose cyclicpeptides that are fibrinogen receptor antagonists.

Barker et al., 1991, PCT/US90/03788 disclose cyclic peptides forinhibiting platelet aggregation.

Pierschbacher et al., 1991, PCT/US91/02356 disclose cyclic peptides thatare fibrinogen receptor antagonists.

Duggan et al, 1992, European Patent Application 92304111.5 disclosefibrinogen receptor antagonists.

Garland et al, 1992 European Patent Applications 92103861.8 and92108214.5 disclose phenylamide derivatives as platelet aggregationinhibitors.

Bondinell et al, 1993, International Patent Application Serial No.PCT/US92/05463 disclose bicyclic fibrinogen antagonists.

Blackburn et al., International Patent Application Serial No.PCT/US92/08788, disclose nonpeptidyl integrin inhibitors havingspecificity for the GPIIb/IIIa receptor.

Egbertson et al., 1992, European Patent Application 0478328A1 disclosetyrosine derivatives that bind with high affinity to the GPIIb/IIIareceptor.

Ojima et al., 1992, 204th Meeting, Amer. Chem. Soc. Abst. 44 disclosesynthetic multimeric RDGF peptides useful in inhibiting plateletaggregation.

Hartman et al., 1992, J. Med. Chem. 35: 4640-4642 describe tyrosinederivatives that have a high affinity for the GPIIb/IIIa receptor.

Radiolabeled peptides for radioimaging thrombi have been reported in theprior art.

Stuttle, 1990, PCT/GB90/00933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

The use of chelating agents for radiolabeling peptides, and methods forlabeling peptides with Tc-99m are known in the prior art and aredisclosed in co-pending U.S. patent applications Ser. No. 07/653,012,now abandoned, which was allowed as U.S. Pat. No. 5,654,272; Ser. No.07/807,062, now U.S. Pat. No. 5,443,815; Ser. Nos. 07/871,282;07/886,752, now abandoned, which has been allowed as U.S. Pat. No.5,736,122; Ser. No. 07/893,981, now U.S. Pat. No. 5,508,020; Ser. No.07/955,466, now abandoned; Ser. No. 08/019,864, now U.S. Pat. No.5,552,525; Ser. No. 08/073,577, now U.S. Pat. No. 5,561,220; Ser. No.08/210,822, now abandoned; Ser. No. 08/236,402 and Ser. No. 08/241,625,now U.S. Pat. No. 5,783,170, and radiolabeled peptides for use asscintigraphic imaging agents for imaging thrombi are known in the priorart and are disclosed in co-pending U.S. patent applications Ser. Nos.07/886,752, 07/893,981 and 08/044,825, which issued as U.S. Pat. No.5,645,815 and International Patent Applications Serial Nos.PCT/US92/00757, PCT/US92/10716, PCT/US93/02320, PCT/US93/03687,PCT/US93/04794, PCT/US93/05372, PCT/US93/06029, PCT/US93/09387,PCT/US94/01894, PCT/US94/03878, and PCT/US94/05895, each of which arehereby incorporated by reference in its entirety.

There remains a need for small (to enhance blood and background tissueclearance), synthetic (to make routine manufacture practicable and toease regulatory acceptance), high-affinity, specific-binding moleculesradiolabeled with a convenient radiolabel, preferably Tc-99m, for use inimaging thrombi in vivo. Small synthetic compounds that bindspecifically to the GPIlb/IIIa receptor on activated platelets, that areradiolabeled with a convenient radioisotope, preferably Tc-99m, ¹¹¹ Inor ⁶⁸ Ga, fulfill this need in the art, and are provided by thisinvention.

SUMMARY OF THE INVENTION

This invention provides small, synthetic, radiolabeled (preferablyTc-99m, ¹¹¹ In or ⁶⁸ Ga labeled) compounds that bind to the GPIlb/IIIareceptor with high affinity, as scintigraphic agents for non-invasiveimaging of thrombi in vivo. The invention thereby provides scintigraphicthrombus imaging agents that are radioactively-labeled reagents.Specifically, the invention provides reagents for preparing thrombusimaging agents that are radiolabeled with technetium-99m (Tc-99m), ¹¹¹In or ⁶⁸ Ga, preferably with Tc-99m. The reagents of the invention areeach comprised of a specific binding compound, including but not limitedto peptides, that binds specifically and with high affinity to theplatelet glycoprotein IIb/IIIa (GPIIa/IIIb) receptor, and is covalentlylinked to a radiolabel-complexing moiety.

We have found that, for optimal imaging, the reagent must be capable ofbinding to the platelet GPIIb/IIIa receptor with sufficient affinitythat it inhibits the adenosine diphosphate (ADP)-induced aggregation ofhuman platelets in a standard platelet aggregation assay (see Example 3below) to the extent of 50% when present at a concentration of no morethan 1 μM.

It is of distinct commercial advantage to use small compounds,preferably having a molecular weight of less than about 10,000 daltons.Such small compounds can be readily manufactured. Moreover, they arelikely not to be immunogenic and to clear rapidly from the vasculature,thus allowing for better and more rapid imaging of thrombi. In contrast,larger molecules such as antibodies of fragments thereof, or otherbiologically-derived peptides larger than 10,000 daltons, are costly tomanufacture, and are likely to be immunogenic and clear more slowly fromthe bloodstream, thereby interfering with rapid diagnoses of thrombi invivo.

The invention also provides reagents wherein the specific bindingcompounds are linear or cyclic peptides having an amino acid sequence of4 to 100 amino acids and a molecular weight no greater than about 10,000daltons.

One aspect of the invention provides a reagent for preparing a thrombusimaging agent that is capable of being radiolabeled for imaging thrombiwithin a mammalian body, comprising a specific binding compound thatspecifically binds to the platelet GPIlb/IIIa receptor, and that iscovalently linked to a Tc-99m complexing moiety of formula:

    C(pgp).sup.S --(aa)--C(pgp).sup.S                          I.

wherein C(pgp)^(S) is a protected cysteine and (aa) is any primary α- orβ-amino acid not containing a thiol group. In a preferred embodiment,the amino acid is glycine.

In another embodiment, the invention provides a reagent for preparing athrombus imaging agent that is capable of being radiolabeled for imagingthrombi within a mammalian body, comprising a specific binding compoundthat specifically binds to the platelet GPIIb/IIIa receptor, that iscovalently linked to a Tc-99m complexing moiety comprising a singlethiol-containing moiety of formula:

    A--CZ(B)--{(C(R.sup.1 R.sup.2)}.sub.n --X                  II.

wherein A is H, HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acidor peptide)-OOC or R⁴ ; B is H, SH or --NHR³, --N(R³)-(amino acid orpeptide) or R⁴ ; Z is H or R⁴ ; X is SH or --NHR³, --N(R³)-(amino acidor peptide) or R⁴ ; R¹, R², R³ and R⁴ are independently H or straight orbranched chain or cyclic lower alkyl; n is 0, 1 or 2; wherein (peptide)is a peptide of 2 to about 10 amino acids; and: (1) where B is --NHR³ or--N(R³)-(amino acid or peptide), X is SH and n is 1 or 2; (2) where X is--NHR³ or --N(R³)-(amino acid or peptide), B is SH and n is 1 or 2; (3)where B is H or R⁴, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC, X is SH and n is 0 or 1; (4) where A is Hor R⁴, then where B is SH, X is --NHR³ or --N(R³)-(amino acid orpeptide) and where X is SH, B is --NHR³ or --N(R³)-(amino acid orpeptide); (5) where X is H or R⁴, A is HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC and B is SH; (6) where Z ismethyl, X is methyl, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC and B is SH and n is 0; and wherein thethiol moiety is in the reduced form and wherein (amino acid) is anyprimary α- or β-amino acid not containing a thiol group.

In particular embodiments of this aspect of the invention, theradiolabel-complexing moiety has a formula that is:

IIa. -(amino acid)¹ -(amino acid)² {A--CZ(B)--{C(R¹ R²)}_(n) --X },

IIb. -{A--CZ(B)--{C(R¹ R² (}_(n) --X}-(amino acid)¹ -(amino acid)²,

IIc. -(a primary α,ω- or β,ω-diamino acid)-(amino acid)¹-{A--CZ(B)--{C(R¹ R²)}_(n) --X}, or

IId. -{A--CZ(B)--{C(R¹ R²)}_(n) --X}-(amino acid)¹ -(a primary α,ω- orβ,ω-diamino acid)

wherein (amino acid)¹ and (amino acid)² are each independently anynaturally-occurring, modified, substituted or altered α- or β-amino acidnot containing a thiol group; A is H, HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC or R⁴ ; B is H, SH or --NHR³,--N(R³)-(amino acid or peptide) or R⁴ ; Z is H or R⁴ ; X is SH or--NHR³, --N(R³)-(amino acid or peptide) or R⁴ ; R¹, R², R³ and R⁴ areindependently H or straight or branched chain or cyclic lower alkyl; nis an integer that is either 0, 1 or 2; (peptide) is a peptide of 2 toabout 10 amino acids; and: (1) where B is --NHR or --N(R 3)-(amino acidor peptide), X is SH and n is 1 or 2; (2) where X is --NHR or--N(R³)-(amino acid or peptide), B is SH and n is 1 or 2; (3) where B isH or R⁴, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acid orpeptide)-OOC, X is SH and n is 0 or 1; (4) where A is H or R⁴, thenwhere B is SH, X is --NHR³ or --N(R³)-(amino acid or peptide) and whereX is SH, B is --NHR³ or --N(R³)-(amino acid or peptide); (5) where X isH or R⁴, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acid orpeptide)-OOC and B is SH; (6) where Z is methyl, X is methyl, A is HOOC,H₂ NOC, (amino acid or peptide)-NHOC, (amino acid or peptide)-OOC and Bis SH and n is 0; and wherein the thiol group is in the reduced form.

In another embodiment, the invention provides a reagent for preparing athrombus imaging agent that is capable of being radiolabeled for imagingthrombi within a mammalian body, comprising a specific binding compoundthat specifically binds to the platelet GPIIb/IIIa receptor, and that iscovalently linked to a radiolabel-complexing moiety of formula: ##STR1##(for purposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolinic acid (Pic)-based moieties);or

specific binding compound ##STR2## (for purposes of this invention,radiolabel-binding moieties having this structure will be referred to aspicolylamine (Pica)-based moieties); wherein X is H or a protectinggroup; (amino acid) is any primary α- or β-amino acid not containing athiol group; the radiolabel-complexing moiety is covalently linked tothe specific binding compound and the complex of theradiolabel-complexing moiety and the radiolabel is electrically neutral.In a preferred embodiment, the amino acid is glycine and X is anacetamidomethyl protecting group. In additional preferred embodiments,the specific binding compound is covalently linked to theradiolabel-complexing moiety via an amino acid, most preferably glycine.

Yet another embodiment of the invention provides a reagent for preparinga thrombus imaging agent that is capable of being radiolabeled forimaging thrombi within a mammalian body, comprising a specific bindingcompound that specifically binds to the platelet GPIIb/IIIa receptor,and that is covalently linked to a radiolabel-complexing moiety that isa bisamino bisthiol radiolabel-complexing moiety. The bisamino bisthiolmoiety in this embodiment of the invention has a formula selected fromthe group consisting of: ##STR3## wherein each R⁵ can be independentlyH, CH₃ or C₂ H₅ ; each (pgp)^(S) can be independently a thiol protectinggroup or H; m, n and p are independently 2 or 3; A is linear or cycliclower alkyl, aryl, heterocyclyl, combinations or substituted derivativesthereof; and X is a specific binding compound; and ##STR4## wherein eachR⁵ is independently H, lower alkyl having 1 to 6 carbon atoms, phenyl,or phenyl substituted with lower alkyl or lower alkoxy; m, n and p areindependently 1 or 2; A is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; V is H orCO-(amino acid or peptide); R⁶ is H, (amino acid) or peptide or aspecific binding compound; provided that when V is H, R⁶ is amino acidor peptide or a specific binding compound and when R⁶ is H, V is aminoacid or peptide or a specific binding compound, wherein (amino acid) isany primary α- or β-amino acid not containing a thiol group. (Forpurposes of this invention, radiolabel-binding moieties having thesestructures will be referred to as "BAT" moieties). In a preferredembodiment, the specific binding compound is covalently linked to theradiolabel-complexing moiety via an amino acid, most preferably glycine.

In preferred embodiments of the aforementioned aspects of thisinvention, the specific binding compound is a peptide is comprised ofbetween 4 and 100 amino acids. The most preferred embodiment of theradiolabel is technetium-99m.

The reagents of the invention may be formed wherein the specific bindingcompounds or the radiolabel-complexing moieties are covalently linked toa polyvalent linking moiety. Polyvalent linking moieties of theinvention are comprised of at least 2 identical linker functional groupscapable of covalently bonding to specific binding compounds orradiolabel-complexing moieties. Preferred linker functional groups areprimary or secondary amines, hydroxyl groups, carboxylic acid groups orthiol-reactive groups. In preferred embodiments, the polyvalent linkingmoieties are comprised of bis-succinimidylmethylether (BSME),4-(2,2-dimethylacetyl)benzoic acid (DMAB), tris(succinimidylethyl)amine(TSEA), tris(acetamidoethyl)amine, bis-(acetamidoethyl)ether,bis-(acetamidomethyl)ether,N-{2-(N',N'-bis(2-succinimidoethyl)aminoethyl)}-N⁶,N⁹-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide (BAT-BS),α,ε-bisacetyllysine, lysine and 1,8-bis-acetamido-3,6-dioxa-octane.

The invention also comprises scintigraphic imaging agents that arecomplexes of the reagents of the invention with Tc-99m, ¹¹¹ In or ⁶⁸ Ga,most preferably Tc-99m and methods for radiolabeling the reagents of theinvention to provide such scintigraphic imaging agents. Tc-99mradiolabeled complexes provided by the invention are formed by reactingthe reagents of the invention with Tc-99m in the presence of a reducingagent. Preferred reducing agents include but are not limited todithionite ion, stannous ion and ferrous ion. Complexes of the inventionare also formed by labeling the reagents of the invention with Tc-99m byligand exchange of a prereduced Tc-99m complex as provided herein.

The invention also provides kits for preparing scintigraphic imagingthat are the reagents of the invention radiolabeled with Tc-99m. Kitsfor labeling the reagents provided by the invention with Tc-99m arecomprised of a sealed vial containing a predetermined quantity of areagent of the invention and a sufficient amount of reducing agent tolabel the reagent with Tc-99m.

This invention provides methods for preparing peptide reagents of theinvention by chemical synthesis in vitro. In a preferred embodiment,peptides are synthesized by solid phase peptide synthesis.

This invention provides methods for using scintigraphic imaging agentsthat are Tc-99m labeled reagents for imaging thrombi within a mammalianbody by obtaining in vivo gamma scintigraphic images. These methodscomprise administering an effective diagnostic amount of Tc-99m labeledreagents of the invention and detecting the gamma radiation emitted bythe Tc-99m label localized at the thrombus site within the mammalianbody.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates scintigraphic imaging of deep-vein thrombi in thethigh in human patients using a Tc-99m radiolabel peptide reagent of theinvention.

FIG. 2 illustrates scintigraphic imaging of deep-vein thrombi in thecalf in human patients using a Tc-99m radiolabel peptide reagent of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides reagents, including peptide reagents, forpreparing radiolabeled thrombus imaging agents for imaging a thrombuswithin a mammalian body. The reagents provided by the invention comprisea radiolabel binding moiety covalently linked to a specific bindingcompound that binds a platelet receptor that is the platelet GPIIb/IIIareceptor and is capable of inhibiting human platelet aggregation inplatelet-rich plasma by 50% when present at a concentration of no morethan 1 μM (i.e., IC₅₀ =1 μM). For purposes of the invention, the termthrombus imaging reagent will refer to embodiments of the inventioncomprising a specific binding compound covalently linked to aradiolabel-complexing moiety and radiolabeled, preferably with Tc-99m,¹¹¹ In or ⁶⁸ Ga, most preferably with Tc-99m.

We have previously found that, for optimal imaging, a reagent asdisclosed herein must be capable of binding to platelet glycoproteinIIb/IIIa receptor with sufficient affinity that it inhibits adenosinediphosphate (ADP)-induced platelet aggregation in a standard assay (seeExample 3) when present at concentrations up to 0.3 μM (IC₅₀ ≦0.3 μM).This invention was disclosed in U.S. patent application Ser. No.08/044,825, now abandoned, which issued as U.S. Pat. No. 5,645,815, andInternational Patent Application Serial No. PCT/US9403878, thedisclosures of both incorporated by reference in their entireties).

We have now found that advantageously high-quality in vivo scintigraphicimages may be obtained using a radiolabeled scintigraphic imaging agentas disclosed herein having an IC₅₀ ≦1 μM. We have found that a dimericreagent of the invention (P280) having an IC₅₀ of 0.087 μM whenunlabeled was converted to its monomeric counterpart (P246) when labeledwith Tc-99m as described herein. This Tc-99m labeled reagent was foundto yield excellent scintigraphic images of venous thrombi in an in vivocanine model system and of deep-vein thrombi in humans in vivo. When themonomer was assayed for platelet aggregation inhibition, the IC₅₀ valuewas found to be 0.85 μM. Thus, these results indicate that embodimentsof the reagents of the invention having IC₅₀ values of up to about 1 μMare useful as efficacious scintigraphic imaging agents.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo-^(99m) Tc generator. Another advantage of thepresent invention is that none of the preferred radionuclides (Tc-99m,Ga-67, In-111) are toxic, in contrast to other radionuclides known inthe art (for example, ¹²⁵ I.

In the Tc-99m complexing moieties and compounds covalently linked tosuch moieties that contain a thiol covalently linked to a thiolprotecting group { (pgp)^(S) } provided by the invention, thethiol-protecting groups may be the same or different and may be but arenot limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

C(CH₃)₃

--9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Preferred protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Each specific-binding peptide-containing embodiment of the invention iscomprised of a sequence of amino acids. The term amino acid as used inthis invention is intended to include all L- and D-, primary α- orβ-amino acids, naturally occurring and otherwise. Specific-bindingpeptides provided by the invention include but are not limited topeptides having the following sequences (the amino acids in thefollowing peptides are L-amino acids except where otherwise indicated):

CH₂ CO.Y_(D).Apc.GDCGGG

CH₂ CO.Y_(D).Apc.GDCKG

CH₂ CO.Y_(D).Apc.GDCGG

CH₂ CO.Y_(D).Apc.GDC

CH₂ CO.Y_(D).Apc.GDCK

CH₂ CO.Y_(D).Amp.GDC

CH₂ CO.Y_(D).Amp.GDCK and

O-(4-piperidinyl)butyl tyrosine.

Specific-binding peptides of the present invention can be chemicallysynthesized in vitro. Peptides of the present invention can generallyadvantageously be prepared on an peptide synthesizer. The peptides ofthis invention can be synthesized wherein the radiolabel-binding moietyis covalently linked to the peptide during chemical synthesis in vitro,using techniques well known to those with skill in the art. Suchpeptides covalently linked to the radiolabel-binding moiety duringsynthesis are advantageous because specific sites of covalent linkagecan be determined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodimentscomprising picolinic acid {(Pic-); e.g., Pic-Gly-Cys(protectinggroup)-}, the radiolabel-binding moiety can be synthesized as the last(i.e., amino-terminal) residue in the synthesis. In addition, thepicolinic acid-containing radiolabel-binding moiety may be covalentlylinked to the ε-amino group of lysine to give, for example,αN(Fmoc)-Lys-εN{Pic-Gly-Cys (protecting group)}, which may beincorporated at any position in the peptide chain. This sequence isparticularly advantageous as it affords an easy mode of incorporationinto the target binding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety{-Cys(protecting group)-Gly-Pica} can be prepared during peptidesynthesis by including the sequence {-Cys(Cys(protecting group)-Gly-} atthe carboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys- and-Cys-Gly-Pica chelators are provided in the Examples hereinbelow. Thisinvention provides for the incorporation of these chelators intovirtually any peptide capable of specifically binding to a thrombus invivo, resulting in a radiolabeled peptide having Tc-99m held as neutralcomplex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m. This invention provides for the incorporation of thesechelators into virtually any peptide capable of specifically binding toa thrombus in vivo, resulting in a radiolabeled peptide having Tc-99mheld as neutral complex. An example of a small synthetic peptidecontaining a BAT chelator as radiolabel-binding moiety is provided inthe Examples hereinbelow.

In forming a complex of radioactive technetium with the reagents of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the reagent in the presence of a reducingagent. Preferred reducing agents are dithionite, stannous and ferrousions; the most preferred reducing agent is stannous chloride. Means forpreparing such complexes are conveniently provided in a kit formcomprising a sealed vial containing a predetermined quantity of areagent of the invention to be labeled and a sufficient amount ofreducing agent to label the reagent with Tc-99m. Alternatively, thecomplex may be formed by reacting a reagent of this invention with apre-formed labile complex of technetium and another compound known as atransfer ligand. This process is known as ligand exchange and is wellknown to those skilled in the art. The labile complex may be formedusing such transfer ligands as tartrate, citrate, gluconate or mannitol,for example. Among the Tc-99m pertechnetate salts useful with thepresent invention are included the alkali metal salts such as the sodiumsalt, or ammonium salts or lower alkyl ammonium salts. In a preferredembodiment of the invention, a kit for preparing technetium-labelledreagents is provided. An appropriate amount of the reagent is introducedinto a vial containing a reducing agent, such as stannous chloride, inan amount sufficient to label the reagent with Tc-99m. An appropriateamount of a transfer ligand as described (such as tartrate, citrate,gluconate or mannitol, for example) can also be included. The kit mayalso contain conventional pharmaceutical adjunct materials such as, forexample, pharmaceutically acceptable salts to adjust the osmoticpressure, buffers, preservatives and the like. The components of the kitmay be in liquid, frozen or dry form. In a preferred embodiment, kitcomponents are provided in lyophilized form.

Radiolabeled thrombus imaging reagents according to the presentinvention may be prepared by the addition of an appropriate amount ofTc-99m or Tc-99m complex into the vials and reaction under conditionsdescribed in Example 4 hereinbelow.

Radioactively-labeled scintigraphic imaging agents provided by thepresent invention are provided having a suitable amount ofradioactivity. In forming Tc-99m radioactive complexes, it is generallypreferred to form radioactive complexes in solutions containingradioactivity at concentrations of from about 0.01 millicurie (mCi) to100 mCi per mL.

The thrombus imaging reagents provided by the present invention can beused for visualizing thrombi in a mammalian body when Tc-99m labeled. Inaccordance with this invention, the Tc-99m labeled reagents areadministered in a single unit injectable dose. The Tc-99m labeledreagents provided by the invention may be administered intravenously inany conventional medium for intravenous injection such as an aqueoussaline medium, or in blood plasma medium. Generally, the unit dose to beadministered has a radioactivity of about 0.01 mCi to about 100 mCi,preferably 1 mCi to 20 mCi. The solution to be injected at unit dosageis from about 0.01 mL to about 10 mL. After intravenous administration,imaging of the thrombus in vivo can take place in a matter of a fewminutes. However, imaging can take place, if desired, in hours or evenlonger, after the radiolabeled peptide is injected into a patient. Inmost instances, a sufficient amount of the administered dose willaccumulate in the area to be imaged within about 0.1 of an hour topermit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

It will also be recognized by those having skill in the relevant artsthat thrombi are commonly found at sites of atherosclerotic plaque; thatintegrin receptors that may bind to the scintigraphic imaging agents ofthe invention may be found in certain tumors; and that such integrinreceptors are involved in cell adhesion processes that accompany orinitiate leukocyte localization at sites of infection. Therefore it willbe recognized that the scintigraphic imaging agents of this inventionhave additional utility as imaging agents for imaging sites in which theGPIIb/IIIa receptor is expressed, including atherosclerotic plaques,tumors and sites of infection.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethylpolystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid or 50/50 trifluoroaceticacid/dichloromethane, optionally containing water, thioanisole,ethanedithiol, and triethylsilane, prepared in ratios of 100:5:5:2.5:2for 1.5-3 h at room temperature.

Where appropriate, N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. For preparingbranched-chain peptide reagents involving peptide chain synthesis fromboth the α- and ε-amines of lysine, Nα(Fmoc)Nε(Fmoc)-lysine was usedduring SPPS. Where appropriate, 2-chloroacetyl and 2-bromoacetyl groupswere introduced either by using the appropriate 2-halo-acetic acid asthe last residue to be coupled during SPPS or by treating the N-terminusfree amino peptide bound to the resin with either 2-halo-aceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP or2-halo-acetic anhydride/diisopropylethylamine in NMP. Where appropriate,HPLC-purified 2-haloacetylated peptides were cyclized by stirring in a0.1-1.0 mg/mL solution at pH8 optionally containing phosphate,bicarbonate or 0.5-1.0 mM EDTA for 0.5-48 hours, followed byacidification with acetic acid, lyophilization and HPLC purification.Where appropriate, Cys-Cys disulfide bond cyclizations were performed bytreating the precursor cysteine-free thiol peptides at 0.1mg/mL in pH 7buffer with aliquots of 0.006 M K₃ Fe(CN)₆ until a stable yellow colorpersisted. The excess oxidant was reduced with excess cysteine, themixture was lyophilized and then purified by HPLC.

Where appropriate, peptide thiol-chloroacetyl derived sulfides wereprepared by reacting single thiol-containing peptides at a concentrationof 2 to 50 mg/mL in water and acetonitrile or THF or DMF at pH 10 withthe appropriate number (e.g., 0.5 molar equivalents for preparing dimersand 0.33 molar equivalents for preparing trimers) of the chloroacetylpolyvalent linker moiety for 0.5 to 24 hours. The solution was thenneutralized with acetic acid, evaporated to dryness, and, if necessary,deprotected using 10 mL TFA and scavengers such as 0.2 mL triethylsilanefor 30 to 90 minutes. The solution was concentrated and the product wasprecipitated with ether. Products were purified by preparative HPLC.

Where appropriate, BSME adducts were prepared by reacting singlethiol-containing peptides (5 to 50 mg/mL in 50 mM sodium phosphatebuffer, pH 7 to 8) with 0.5 molar equivalents of BMME(bis-maleimidomethylether) pre-dissolved in acetonitrile at roomtemperature for approximately 1-18 hours. The solution was concentratedand the product was purified by HPLC.

Where appropriate, TSEA adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 10 to 100 mg/mL peptidein DMF, or 5 to 50 mg/mL peptide in 50 mM sodium phosphate (pH8)/acetonitrile or THF) with 0.33 molar equivalents of TMEA(tris(2-maleimidoethyl)amine; as disclosed in U.S. Ser. No. 08/044,825,incorporated by reference) pre-dissolved in acetonitrile or DMF, with orwithout 1 molar equivalent of triethanolamine, at room temperature forapproximately 1-18 h. Such reaction mixtures containing adducts wereconcentrated and the adducts were then purified using HPLC.

Where appropriate, BAT-BS adducts were prepared by reacting singlethiol-containing peptide (at concentrations of 2 to 50 mg/mL peptide in50 mM sodium phosphate (pH 8)/acetonitrile or THF) with 0.5 molarequivalents of BAT-BM (N-{2-(N'N'-bis(2-maleimido-ethyl)aminoethyl)}-N⁹-(t-butoxycarbonyl)-N⁶,N⁹-bis(2-methyl-2-triphenylmethylthiopropyl)-6,9-diazanonanamide; asdisclosed in U.S. Ser. No. 08/044,825, incorporated by reference)pre-dissolved in acetonitrile or THF, at room temperature forapproximately 1-18 h. The solution was then evaporated to dryness and(BAT-BS)-peptide conjugates deprotected by treatment with 10 mL TFA and0.2 mL triethylsilane for 1 h. The solution was concentrated, theproduct adducts precipitated with ether, and then purified by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS) or electrospray massspectroscopy (ESMS).

EXAMPLE 2 A General Method for Radiolabeling with Tc-99m

A 0.1 mg sample of a peptide reagent prepared as in Example 2 wasdissolved in 0. 1 mL of water, 50 mM potassium phosphate buffer, 0.1 Mbicarbonate buffer or 10% hydroxypropylcyclodextrin (HPCD), each bufferat pH of 5-10. Tc-99m gluceptate was prepared by reconstituting aGlucoscan vial (E.I. DuPont de Nemours, Inc., Wilmington, Del.) with 1.0mL of Tc-99m sodium pertechnetate containing up to 200 mCi and allowedto stand for 15 minutes at room temperature. 25 μL of Tc-99m gluceptatewas then added to the peptide and the reaction allowed to proceed atroom temperature or at 100° C. for 5-30 min and then filtered through a0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using thefollowing conditions: a Waters DeltaPure RP-18, 5 μ, 150 mm×3.9 mmanalytical column was loaded with each radiolabeled peptide and thepeptides eluted at a solvent flow rate equal to 1 mL/min. Gradientelution was performed beginning with 10% solvent A (0.1% CF3COOH/H₂ O)to 40% solvent B₉₀ (0.1 % CF₃ COOH/90% CH₃ CN/H₂ O) over the course of20 min. The Tc-99m labeled peptide purity was determined by HPLC usingthe conditions described in the Footnotes in Table I. Radioactivecomponents were detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 1 using the method described herein.

                                      TABLE I                                     __________________________________________________________________________                                          FABMS                                                                             Radiochernical                                                                         HPLC                       Peptides                              MH.sup.+                                                                          Yield(%)*                                                                              R.sub.T (min)**            __________________________________________________________________________    CH.sub.2 CO.Y.sub.DRGDCC.sub.Acm GC.sub.Acm amide.sup.b                                                             1057                                                                              97.sup.2 10.0, 10.4, 10.6.sup.2     CH.sub.2 CO.Y.sub.D RGDCGGC.sub.Acm GC.sub.Acm amide                                                                1171                                                                              99.sup.2 13.5.sup.2                 CH.sub.2 CO.Y.sub.D.Apc.GDCGGGC.sub.Acm GC.sub.Acm amide                                                            1233                                                                              100.sup.4                                                                              17.1, 18.1.sup.2           GRGDVRGDFKC.sub.Acm GC.sub.Acm amide(SEQ ID NO:3)                                                                   1510                                                                              97.sup.2 16.2, 16.8.sup.2           GRGDVRGDFC.sub.Acm GC.sub.Acm amide(SEQ ID NO:4)                                                                    1382                                                                              94.sup.2 16.4.sup.2                 CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGF.sub.D PRPG.NH.sub.2                                           1845                                                                              90.sup.4 16.6, 16.9.sup.2           (CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2                                                  3020.sup.a                                                                       98.sup.4 9.3.sup.2                  (CH.sub.2 CO.Y.sub.D.Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.3                                                 4596A                                                                             99.sup.4 9.2, 11.6.sup.5            (CH.sub.2 CO.Y.sub.D Apc.GDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2           -(BAT-BS)                              3409.sup.a                                                                       98.sup.3 10.3.sup.5                 C.sub.Acm GC.sub.Acm RRRRRRRRRGDV(SEQ ID NO:5)                                                                      2100                                                                              100.sup.2                                                                              2.4.sup.3 ***              (CH.sub.2 CO.Y.sub.D Apc.GDCKGC.sub.Acm GC.sub.Acm GGC.amide).sub.2                                                  3163.sup.a                                                                       98.sup.4 9.6.sup.5                  (CH.sub.2 COY.sub.D.Amp.GDCGGC.sub.Acm GC.sub.Acm GGCamide).sub.2             -(CH.sub.2 CO).sub.2 K(Nε-K)GCamide                                                                          3357.sup.a                                                                       99.sup.8 4.6.sup.6                  (CH.sub.2 COY.sub.D.Amp.GDCKGCGamide).sub.2 -(CH.sub.2 CO).sub.2 K(N.epsil    on.-K)GCamide                          2573.sup.a                                                                       99.sup.8 4.8.sup.6                  (CH.sub.2 COY.sub.D.Apc.GDCGGC.sub.Acm GC.sub.Acm GGCamide).sub.2             (CH.sub.2 CO).sub.2 -K(Nε-K)GCamide                                                                          3298.sup.a                                                                       96.sup.3 12.0.sup.4                 __________________________________________________________________________    *Superscripts refer to the following labeling conditions:                       The peptide was dissolved in 50 mM potassium phosphate buffer (pH 7.4)        and labeled at room                                                           temperature.                                                                  The peptide was dissolved in 50 mM potassium phosphate buffer (pH 7.4)        and labeled at 100° C.                                                 The peptide was dissolved in water and labeled at room temperature.           The peptide was disso1ved in water and labeled at 100° C.              The peptide was dissolved in 50 mM potassium phosphate buffer (pH 6.0)        and labeled at 100° C.                                                 The peptide was dissolved in 50 mM potassium phosphate buffer (pH 5.0)        and labeled at room                                                           temperature.                                                                  The peptide was dissolved in a 50:50 mixture of ethano1/water and             labeled at 100° C.                                                     The peptide was dissolved in 0.9% sodium chloride solution and labeled        at room temperature.                                                        **HPLC methods (indicated by superscript after RT):                           general:                                                                            solvent A = 0.1% CF.sub.3 COOH/H.sub.2 O                                      solvent B.sub.70  =                                                                       0.1% CF.sub.3 COOH/70% CH.sub.3 CN/H.sub.2 O                      solvent B.sub.90  =                                                                       0.1% CF.sub.3 COOH/90% CH.sub.3 CN/H.sub.2 O                      solvent flow rate =                                                                       1 mL/min                                                    Vydak column = Vydak 218TP54 RP-18, 5 μm, 220 mm × 4.6 mm            analytical column with guard column                                           Brownlee column = Brownlee Spheri-5 RP-18, 5 μm, 220 mm × 4.6 mm     column                                                                        Waters column = Waters Delta-Pak C18, 5 μm, 150 mm × 3.9 mm          column                                                                        Waters column 2 = Waters Nova-Pak C18, 5μm, l00 mm × 8 mm radial     compression column                                                            Method 1:                                                                           Brownlee column                                                                         100% A to 100% B.sub.70  in 10 min                            Method 2:                                                                           Vydak column                                                                            100% A to 100% B.sub.90  in 10 min                            Method 3:                                                                           Vydak column                                                                            100% A to 100% B.sub.70  in 10 min                            Method 4:                                                                           Waters column                                                                           100% A to 100% B.sub.90  in 20 min                            Method 5:                                                                           Waters column                                                                           100% A to 100% B.sub.90  in 10 min                            Method 6:                                                                           Waters 2 column                                                                         100% A to 100% B.sub.90  in 10 min                            ***Confirmed by sodium dodecyl sulfate-polyacrylamide gel                     electrophoresis                                                               Single-letter abbreviations for amino acids can be found in G. Zubay,         Biochemistry (2d. ed.), 1988                                                  (MacMillen Publishing: New York) p.33; underlining indicates the              formation of a thiol linkage                                                  between the linked amino acids of derivative groups; peptides are linked      to BSH, ETAC, BSME,                                                           TSEA, (BAT-BS) or (CH.sub.2 CO)-containing linkers via the free thiol         moiety of the unprotected                                                     cysteine residue (C) in each peptide; Ac = acetyl; Bz = benzoyl; Pic =        picolinoyl                                                                    (pyridine-2-carbonyl); Acm = acetamidomethyl; Mob = 4-methoxybenzyl; Apc      = L-(S-(3-                                                                    aminopropyl)cysteine); Hly = homolysine; FD = D-phenylalanine; YD =           D-tyrosine; ma = 2-                                                           mercaptoacetic acid; mmp = 2-mercapto-2-methylpropionic acid; BAT =           N.sup.6,N.sup.9 -bis(2-mercapto-2-                                            methylpropyl)-6,9-diazanonanoic acid; ETAC = 4-(O--CH.sub.2 CO--Gly--Gly--    Cys.amide)acetophenone;                                                       BAT-BS = N-{2-N',N'-bis(2-succinimidoethyl) aminoethyl }-N.sup.6,N.sup.9      -bis(2-mercapto-2-                                                            methylpropyl)-6,9-diazanonanamide; BSME = bis-succinimidomethylether;         TSEA = tris-(2-                                                               succinimidoethyl)amine; NES = N-ethylsuccinimide; .BSH = 1,6-bis-succinimi    dohexane; Amp = 4-                                                            amidinophenylalanine                                                          .sup.a  = confirmed by electrospray mass spectrometry (ESMS)              

EXAMPLE 3 Platelet Aggregation Inhibition Assays

Platelet aggregation studies were performed essentially as described byZucker (1989, Methods in Enzymol. 169: 117-133). Briefly, plateletaggregation was assayed with or without putative platelet aggregationinhibitory compounds using fresh human platelet-rich plasma, comprising300,000 platelets per microliter. Platelet aggregation was induced bythe addition of a solution of adenosine diphosphate to a finalconcentration of 10 to 15 micromolar, and the extent of plateletaggregation monitored using a Bio/Data aggregometer (Bio/Data Corp.,Horsham, Pa.). The concentrations of platelet aggregation inhibitorycompounds used were varied from 0.1 to 500 μg/mL. The concentration ofinhibitor that reduced the extent of platelet aggregation by 50%(defined as the IC₅₀) was determined from plots of inhibitorconcentration versus extent of platelet aggregation. An inhibition curvefor peptide RGDS was determined for each batch of platelets tested.

The results of these experiments are shown in Table II. In Table II, thecompounds tested are as follows (RGDS is given as a positive control):

P47=AcSYGRGDVRGDFKC_(Acm) GC_(Acm) (SEQ ID NO: 6)

P97=GRGDVRGDFKC_(Acm) GC_(Acm) amide (SEQ ID NO: 3)

P32=C_(Acm) GC_(Acm) RRRRRRRRRGDV (SEQ ID NO: 5)

P143=CH₂ CO--Y_(D) RGDCGGC_(Acm) GC_(Acm) amide

P245=CH₂ CO--Y_(D).Apc.GDCGGC_(Acm) GC_(Acm) GGF_(D) PRPGamide

P63=AcSYGRGDVRGDFKCTCCA (SEQ ID NO: 7)

P98=GRDGVRGDFC_(Acm) GC_(Acm) amide(SEQ ID NO: 8)

P81=CH₂ CO--Y_(D) RGDCC_(Acm) GC_(Acm) amide

P154=CH₂ CO--Y_(D) ApcGDCGGGC_(Am) GC_(Acm) amide

P381=(CH₂ CO--Y_(D) ApcGDCKGC_(Acm) GC_(Acm) GGC-amide)₂ -BSME

P317=(CH₂ CO--Y_(D) ApcGDCGGC_(Acm) GC_(Acm) GGC-amide)₃ -TSEA

P246=CH₂ CO--Y_(D) ApcGDCGGC_(Acm) GC_(Acm) GGC-amide

P357=(CH₂ CO--Y_(D) ApcGDCGGC_(Acm) GC_(Acm) GGC-amide)₂ -(BAT-BS)

P667=(CH₂ COY_(D).Apc.GDCGGC_(Acm) GC_(Acm) GGCamide)₂ (CH₂ CO)₂K(Nε-K)GCamide

P747=(CH₂ COY_(D).Amp.GDCGGC_(Acm) GC_(Acm) GGCamide)₂ (CH₂ CO)₂K(Nε-K)GCamide

P748=(CH₂ COY_(D).Amp.GDCKGCGamide)₂ (CH₂ CO)₂ K(Nε-K)GCamide

(Single-letter abbreviations for amino acids can be found in G. Zubay,Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p.33;Ac=acetyl; Acm=acetamidomethyl; Apc=L-(S-(3-aminopropyl)cysteine); Y_(D)=D-tyrosine; BSME=bis-succinimidylmethylether;TSEA=tris(succinimidylethyl)amine;(BAT-BS)=N-{2-(N',N'bis(2-succinimidoethyl) aminoethyl)}-N⁶,N⁹,-bis(2-methyl-2-mercaptopropyl)-6,9-diazanonanamide; peptides arelinked to BSME, TSEA, (BAT-BS) or (CH₂ CO)-containing linkers via thefree thiol moiety of the unprotected cysteine residue (C) in eachpeptide). (. . . )₂ K represents a covalent bond between the moiety inparenthesis and each of the amino groups (i.e., the α-amino and thesidechain amine) of lysine. (Nε-K) represents covalent linkage at the εamine rather than at the usual α amino group of the lysine residue.

                  TABLE II                                                        ______________________________________                                        Peptides     IC.sub.50 (μM)**                                                                     Clot/Blood*                                            ______________________________________                                        P357         0.079     6.3 ± 3.4.sup.5                                     P667         0.081     5.9, 5.0.sup.2                                         P682         0.130      4.0.sup.1                                             P317         0.036     3.8 ± 2.2.sup.3                                     P381         0.035     2.5                                                    P154         0.30      2.0 ± 0.5.sup.3                                     P246         0.85      4.4 ± 1.8                                           P143         1.3       1.4                                                    P97          8         1.0                                                    P98          15        1.7                                                    P63          19        1.7                                                    P47          23        1.0                                                    P81          25        1.8 ± 0.6.sup.3                                     P32          26        1.2 ± 0.2.sup.4                                     ______________________________________                                         .sup.1 n = 1;                                                                 .sup.2 n = 2;                                                                 .sup.2 n = 3;                                                                 .sup.4 n = 4;                                                                 .sup.5 n = 9                                                                  *ratio of (% injected dose/g in a femoral vein thrombus)/(% injected          dose/g in blood) at approximately 4 h postinjection of each Tc99 m labele     reagent in a canine model of DVT                                              **concentration of reagent that inhibits by 50% the aggregation of human      platelets in plateletrich plasma induced to aggregate by the addition of      adenosine diphosphate (ADP)                                              

These results demonstrate that the compounds having an IC₅₀ less than orequal to about 1 μM show greater efficacy as scintigraphic imagingagents that compounds having an IC₅₀ greater than about 1 μM.

EXAMPLE 4

Scintigraphic Imaging of Deep-Vein Thrombi in Humans in vivo

A series of experiments comprising a pilot human clinical study of oneembodiment of the scintigraphic imaging agents of the invention,designated P280, having the chemical structure:

    (CH.sub.2 CO.Y.sub.D ApcGDCGGC.sub.Acm GC.sub.Acm GGC.amide).sub.2 -BSME

were performed. These experiments were performed on 9 human patients (6males, 3 females), ages 30 to 60 years and weighing 63 to 100 kg. Eachof the patients presented clinically with the symptoms of deep-veinthrombosis, and the diagnosis was confirmed by physical work-up,ultrasonography and/or contrast venography.

Each patient in the study was administered 10-22 mCi Tc-99m labeled P280comprising approximately 0.25 mg peptide, by intravenous injection.Scintigraphic imaging was then performed over four hours using a largefield-of-view gamma camera equipped with a high-resolution collimator(photopeaked at 140 keV, with a 20%±window). Gamma camera imaging wascommenced simultaneously with injection. Anterior images followed byposterior images over the legs were acquired over the first hour, thenat 2 and 4 hours post-injection.

The results of these studies are shown in FIG. 1, which shows thrombuslocalization in vessels of the lower thigh and FIG. 2, which showsthrombus localization in vessels of the calf. Thrombi so localized arehighlighted with an arrow in each Figure. In addition to rapidly andefficiently localizing sites of deep-vein thrombi, this scintigraphicimaging agent was found to clear rapidly from the bloodstream, resultingin less than about 10% of the injected dose remaining in the circulation1 hour post-injection. Consistent with animal studies, at least 60-70%of the injected dose was found to be cleared by the kidneys. Thrombusvisualization was evident as early as 15-30 min after injection, andremained visible for 4-24 hours post-injection. Thrombus visualizationcorrelated well with the clinical diagnosis of deep-vein thrombosis madebased on the aforementioned conventional clinical criteria, thrombibeing visualized in 8 of the 9 patients studied. The one patient inwhich a thrombus was not visualized presented with a clinically oldthrombus (42 days), which was likely quiescent and hence no longerexperiencing platelet turnover at its surface. Finally, no toxicity orother adverse effects of Tc-99m labeled P280 administration wereobserved in any of these patients.

These results demonstrate that the scintigraphic imaging agents of theinvention represents a safe and effective diagnostic reagent forpreparing clinically-effective scintigraphic imaging agents useful forclinical, in vivo use for visualizing deep-vein thrombi in humans.

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - <160> NUMBER OF SEQ ID NOS: 8                                               - <210> SEQ ID NO 1                                                           <211> LENGTH: 6                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 <220> FEATURE:                                                                #an amino acid or if Xaa Xaa at position 1 is                                       is not                                                                  #be attached to theTION: present, a hydrogen will                                   adjacent                                                                <223> OTHER INFORMATION: residue.                                             <220> FEATURE:                                                                #be Thr or CysFORMATION: Xaa at position 5 may                                <220> FEATURE:                                                                #an amino acid, or if XaaXaa at position 6 is                                       is not                                                                  #be attached to theTION: present, a hydrogen will                                   adjacent                                                                <223> OTHER INFORMATION: residue.                                             - <400> SEQUENCE: 1                                                           - Xaa Arg Gly Asp Xaa Xaa                                                       1               5                                                           - <210> SEQ ID NO 2                                                           <211> LENGTH: 4                                                               <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 <220> FEATURE:                                                                #be Ser, Thr, or CysION: Xaa at position 4 may                                - <400> SEQUENCE: 2                                                           - Arg Gly Asp Xaa                                                               1                                                                           - <210> SEQ ID NO 3                                                           <211> LENGTH: 13                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (11)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (13)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (13)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 3                                                           - Gly Arg Gly Asp Val Arg Gly Asp Phe Lys Cy - #s Gly Cys                     #                10                                                           - <210> SEQ ID NO 4                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (10)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 4                                                           - Gly Arg Gly Asp Val Arg Gly Asp Phe Cys Gl - #y Cys                         #                 10                                                          - <210> SEQ ID NO 5                                                           <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (3)                                                           <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 5                                                           - Cys Gly Cys Arg Arg Arg Arg Arg Arg Arg Ar - #g Arg Gly Asp Val             #                 15                                                          - <210> SEQ ID NO 6                                                           <211> LENGTH: 15                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: ACETYLATION                                          <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (13)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (15)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 6                                                           - Ser Tyr Gly Arg Gly Asp Val Arg Gly Asp Ph - #e Lys Cys Gly Cys             #                 15                                                          - <210> SEQ ID NO 7                                                           <211> LENGTH: 17                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (1)                                                           <223> OTHER INFORMATION: ACETYLATION                                          <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 7                                                           - Ser Tyr Gly Arg Gly Asp Val Arg Gly Asp Ph - #e Lys Cys Thr Cys Cys         #                 15                                                          - Ala                                                                         - <210> SEQ ID NO 8                                                           <211> LENGTH: 12                                                              <212> TYPE: PRT                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (10)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: acetamidomethyl                                      <220> FEATURE:                                                                <221> NAME/KEY: MOD.sub.-- RES                                                <222> LOCATION: (12)                                                          <223> OTHER INFORMATION: AMIDATION                                            <220> FEATURE:                                                                #Sequence:syntheticTION: Description of Artificial                                  peptide                                                                 - <400> SEQUENCE: 8                                                           - Gly Arg Asp Gly Val Arg Gly Asp Phe Cys Gl - #y Cys                         #                 10                                                          __________________________________________________________________________

What is claimed is:
 1. A thrombus imaging agent comprising:a) a reagentcomprisingi) a radiolabel complexing moiety; and ii) a plateletglycoprotein IIb/IIIa-binding compound covalently linked to the moietyand having a molecular weight less than 10,000 daltons;wherein saidreagent is capable of inhibiting human platelet aggregation inplatelet-rich plasma by 50% when present at a concentration not greaterthan about 1 μM and b) a radiolabel.
 2. The agent of claim 1, whereinthe complexing moiety comprises a single-thiol-containing moiety havinga formula:

    A--CZ(B)--{C(R.sup.1 R.sup.2)}.sub.n --X

wherein A is H, HOOC, H₂ NOC, (amino acid or peptide)-NHOC, (amino acidor peptide)-OOC or R⁴ ; B is H, SH, --NHR³, --N(R³)-(amino acid orpeptide), or R⁴ ; X is H, SH, --NHR³, --N(R³)-(amino acid or peptide) orR⁴ ; Z is H or R⁴ ; R¹, R², R³ and R⁴ are independently H or lowerstraight or branched chain or cyclic alkyl; n is 0, 1 or 2; (peptide) isa peptide having between 2 and about 10 amino acids; andwhere B is--NHR³ or --N(R³)-(amino acid or peptide), X is SH, and n is 1 or 2;where X is --NHR³ or --N(R³)-(amino acid or peptide), B is SH, and n is1 or 2; where B is H or R⁴, A is HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC, X is SH, and n is 0 or 1;where A is H or R⁴, then where B is SH, X is --NHR₃ or --N(R³)-(aminoacid or peptide) and where X is SH, B is --NHR³ or --N(R³)-(amino acidor peptide); where X is H or R⁴, A is HOOC, H₂ NOC, (amino acid orpeptide)-NHOC, (amino acid or peptide)-OOC and B is SH; where Z ismethyl, X is methyl, A is HOOC, H₂ NOC, (amino acid or peptide)-NHOC,(amino acid or peptide)-OOC, B is SH and n is 0; and wherein the thiolmoiety is in the reduced form and (amino acid) is any primary α- orβ-amino acid not containing a thiol group.
 3. The agent of claim 2,wherein the complexing moiety is selected from the group consistingof:-(amino acid)¹ -(amino acid)² -{A--CZ(B)--{C(R¹ R²)}_(n) --X};-{A--CZ(B)--{C(R¹ R²)}_(n) --X}-(amino acid)¹ -(amino acid)² ; -(aprimary α,ω- or β,ω-diamino acid)-(amino acid)¹ -{A--CZ(B)--{C(R¹R²)}_(n) --X}; and -{A--CZ(B)--{C(R¹ R²)}_(n) --X}-(amino acid)¹ -(aprimary α,ω- or β,ω-diamino acid),wherein (amino acid)¹ and (aminoacid)² are each independently any naturally-occurring, modified,substituted or altered α- or β-amino acid not containing a thiol group.4. The agent of claim 1, wherein the reagent is

    CH.sub.2 CO--Y.sub.D ApcGDCGGC.sub.Acm GC.sub.Acm GGC.amide.


5. The agent of claim 1, wherein the radiolabel is selected from thegroup consisting of technetium-99m, indium-111, and gallium-68.
 6. Acomplex formed by reacting technetium-99m and a reducing agent with areagent comprising:a) a radiolabel complexing moiety; and b) a plateletglycoprotein IIb/IIIa-binding compound covalently linked to the moietyand having a molecular weight less than 10,000 daltons;wherein saidreagent is capable of inhibiting human platelet aggregation inplatelet-rich plasma by 50% when present at a concentration not greaterthan about 1 μM.
 7. The complex of claim 6, wherein the reducing agentis selected from the group consisting of a dithionite ion, a stannousion, and a ferrous ion.
 8. A complex formed by labeling a reagentcomprising:a) a radiolabel complexing moiety; and b) a plateletglycoprotein IIb/IIIa-binding compound covalently linked to the moietyand having a molecular weight less than 10,000 daltons;wherein saidreagent is capable of inhibiting human platelet aggregation inplatelet-rich plasma by 50% when present at a concentration not greaterthan about 1 μM; with technetium-99m by ligand exchange of a prereducedtechnetium-99m complex.
 9. A kit comprising a sealed vial containing:a)a predetermined quantity of a reagent comprising:i) a radiolabelcomplexing moiety; and ii) a platelet glycoprotein IIb/IIIa-bindingcompound covalently linked to the moiety and having a molecular weightless than 10,000 daltons;wherein said reagent is capable of inhibitinghuman platelet aggregation in platelet-rich plasma by 50% when presentat a concentration not greater than about 1 μM; and b) a sufficientamount of a reducing agent to label the reagent with technetium-99m. 10.A method of making the imaging agent of claim 1, comprising the step ofreacting the reagent with Tc-99m in the presence of a reducing agent.11. The method of claim 10, wherein the reducing agent is selected fromthe group consisting of a dithionite ion, a stannous ion, and a ferrousion.